Xenotransplantation will only become clinically feasible once mechanisms of xenograft loss and rejection are better understood. The development of inbred miniature GalT-KO swine with removal of the dominant xeno-antigen has been a major advance. However, problems still persist in generating mixed xenogeneic chimerism in baboons and obtaining tolerance to xenogeneic cells and vascularized xenografts. Inflammatory reactions to porcine bone marrow-derived cells and the vasculature of organ grafts are linked to aberrant immune responses, together with procoagulant activation and the development of xenograft microvascular injury. Thrombotic processes and the progressive xenograft microangiopathy appear exacerbated by dysfunctional immune reactions and already documented intrinsic molecular incompatibilities in thromboregulation between discordant species. We have made significant progress in defining mechanisms of consumptive coagulopathy linked to associated functional incompatibilities of CD39 and thrombomodulin across species, and by determining novel vascular markers of injury associated with humoral rejection. We have recently shown that CD39 is expressed by the endothelium and also by T regulatory cells. Hence, adenosine generated by this ectonucleotidase blocks platelet activation impeding coagulation and also serves as an immune suppressive mediator. Our new studies will build on these successes and insights to address the following Specific Aims: #1: Study how cellular immune mechanisms and adenosine production by CD39 expressed by T regulatory cells impact xenogeneic tolerance mechanisms (with models developed in Project 3); and #2: Demonstrate therapeutic potential of transgenic upregulation of human CD39 and thrombomodulin in GalT-KO swine in thymokidney xenotransplant and tolerance models (in Projects 1 and 2). These strategies will include optimal immunosuppressive interventions with protocols to attempt induction of tolerance and ameliorate vascular inflammation in baboons. The overall goals of this application will be to effectively manage immune reactions, graft vascular Injury and thrombosis, currently associated with GalT-KO pig-to-baboon thymokidney xenotransplantation, and thereby provide clinically relevant survival times.